Two non-identical heads of myosin molecules.

Four different preparations of skeletal subfragment-1, denoted in this report as S1(Aa), S1(Ab), S1(Ba), and S1(Bb), and two different preparations of cardiac subfragment-1, denoted as S1(A) and S1(B), were obtained as described in our recent report (J. Biochem. 97, 965, 1985). (i) The four preparations were obtained from chicken breast myosin trinitrophenylated with 2,4,6-trinitrobenzene sulfonate in the absence of inorganic pyrophosphate (-PPi), and they were all shown to be trinitrophenylated. Addition of PPi caused change in the absorption spectra of trinitrophenyl(TNP)-S1(Aa) and TNP-S1(Ba), but not in those of TNP-S1(Ab) and TNP-S1(Bb). (ii) The two preparations of S1 were obtained from cardiac myosin trinitrophenylated either in the absence (-) or presence (+) of PPi. S1(B) was trinitrophenylated, whereas S1(A) was not. Specifically emphasized is the observation that the yield of cardiac S1(A) was practically equal to that of cardiac S1(B). On the basis of these results, we propose the hypothesis of "two iso-myosins with non-identical heads," which is essentially a combination of the hypothesis of isoenzymes and that of non-identical heads.

Production of nitrite ions from trinitrophenyl myosin and from trinitrophenyl subfragment-1.

When trinitrophenyl (TNP) myosin of either chicken breast muscle or porcine cardiac muscle was left to stand in an alkaline medium at 20 degrees C for several hours, nitrite ions were found to be gradually produced. The nitrite production from myosin trinitrophenylated in the presence of PPi occurred at the same rate and to the same extent as that from myosin trinitrophenylated in the absence of PPi. The nitrite production was significantly reduced when thiols of myosin were modified with 2-nitro-5-thiocyanobenzoate. Four different preparations of TNP subfragment-1, S1(Aa), S1(Ab), S1(Ba), and S1(Bb), were obtained from chymotryptic digest of chicken breast myosin trinitrophenylated in the absence of PPi. When these preparations of TNP-S1 were left to stand at alkaline pH, a significant amount of nitrite was produced from S1(Ab) and S1(Bb), but very little from S1(Aa) and S1(Ba). In our previous report (J. Biochem. 97, 965-968, 1985), S1(Aa) and S1(Ba) were suggested to correspond to "non-burst" heads of myosin, and S1(Ab) and S1(Bb) to "burst" heads of the myosin molecule (Inoue et al. (1980) Adv. Biophys. 13, 1-194). Therefore, the present findings described above strongly suggest that the nitrite production involves some interaction of TNP groups with thiols, and that it occurs at the "burst" heads.

Two different preparations of subfragment-1 from chicken skeletal myosin and from porcine cardiac myosin.

Two different subfragment-1 preparations were obtained from either skeletal or cardiac myosin. They were identical in the heavy chain and light chain compositions but different in the pH dependence of the Ca-ATPase activity and in the relationship with "reactive lysine residues" (RLR).

Biochemical characteristics of cardiac myosin: the pH dependence of Ca-ATPase activity, and that of the absorption spectrum of 2,4,6-trinitrophenyl groups attached to myosin.

For both cardiac and skeletal myosin, the Ca-ATPase activity of myosin at acidic pH was shown to be different from that at alkaline pH, in the susceptibility to heat-inactivation, the effects of organic solvents, and the effect of trinitrophenylation of the myosin. It is therefore suggested that there are two different types of Ca-ATPase of both cardiac and skeletal myosin. Differences in the Ca-ATPase activity were also found between cardiac and skeletal myosins. (a) The Ca-ATPase activity of cardiac myosin was more susceptible to heat-inactivation at alkaline pH than at acidic pH. In contrast, the activity of skeletal myosin was more susceptible to heat-inactivation at acidic pH than at alkaline pH. (b) Dioxane weakly stimulated the activity of cardiac myosin at acidic pH, but strongly activated that of skeletal myosin at acidic pH. Acetone very strongly inhibited the activity of cardiac myosin at alkaline pH, but not so strongly that of skeletal myosin at alkaline pH. (c) Trinitrophenylation of the myosin resulted in loss of the activity optimum at acidic pH with skeletal myosin but not with cardiac myosin. As reported by Srivastava et al. (J. Biochem. 86, 725-731, 1979), 1 mol of lysine residue per mol of cardiac myosin quickly reacted with 2,4,6-trinitrobenzene sulfonate (TNBS) either in the absence or presence of inorganic pyrophosphate (PPi). However, trinitrophenyl (TNP) groups bound to cardiac myosin in the presence of PPi were significantly different, in the pH dependence of the absorption spectrum, from those bound (to cardiac myosin) in the absence of PPi.(ABSTRACT TRUNCATED AT 250 WORDS)

Plaque assay of equine influenza virus.

ESK cells, a stable cell line derived from a swine embryo kidney, were found to be a good medium for plaque formation of the Prague and Miami strains of equine influenza virus. Factors influencing the plaque formation were investigated and a plaque assay for these viruses was worked out. The method is not only simple enough for routine use, but also is as sensitive as the egg inoculation method. The method was readily adapted for a neutralization test.